1. Technical Field
The invention relates to a method for effecting the conjugative transfer of mobilizable vectors from E. coli into gram-positive bacteria and to vectors suited for this purpose.
2. Background Information
The use of transposons for mutagenesis, gene isolation and gene analysis was, in the past, largely limited to gram-negative bacteria. The mutagenesis of Bacillus subtilis by the transposon Tn917 can be considered an exception in this connection (Youngman et al., PNAS USA 80, 4 (1983), pp. 2305-2309) which is made possible by the fact that B. subtilis has a natural competence for taking up plasmid DNA from the surrounding medium.
P. Trieu-Cuot et al. (FEMS Microbiology Letters 48 (1987), pp. 289-294) describe the plasmid transfer from E. coli into certain gram-positive bacteria by means of conjugation; only very unsatisfactory values are achieved, however, with transfer frequencies of 10.sup.-7 to 10.sup.-8. In addition, Applicants' own tests show that a transfer into Corynebacterium glutamicum cannot be demonstrated with the system described therein.
U.S. Pat. Nos. 4,626,506, 4,680,264 and 4,686,184 (Puhler et al.) and Simon et al. (Biotechnology, November, 1983 and Methods in Enzymology, vol. 118, pp. 640-659) teach the mutagenesis of gram-negative bacteria using mobilizable E. coli vectors. In contrast, the present invention provides a method for effecting the conjugative transfer of mobilizable vectors from E. coli to gram-positive bacteria and vectors suitable for use in same.